Octanoyl Ghrelin Is Hypothalamic Rooted
http://www.100md.com
内分泌学杂志 2005年第6期
Tulane University Health Sciences Center, New Orleans, Louisiana 70112
Address all correspondence and requests for reprints to: Cyril Y. Bowers, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL53, New Orleans, Louisiana 70112. E-mail: cybowers@tulane.edu.
n-Octanoyl ghrelin continues to be noted for its stimulatory action on the GHS-R 1a [GH secretagogue (GHS) receptor] and regulation of GH secretion and food intake (1, 2, 3). This now has been extended more in depth in regard to regulation of food intake as revealed in the publication of Sato and Kojima and co-workers (4). The high-quality, validated approaches and techniques used by the authors to quantitate the molecular forms of hypothalamic ghrelin in vivo in rats is the first reason for concluding that the study is of major importance. At last, unequivocal in vivo evidence has been obtained that n-octanoyl ghrelin exists and is synthesized in the hypothalamus, and that it and des-acyl (des-octanoyl) ghrelin constitute the two major molecular forms of ghrelin in the hypothalamus. The role of des-octanoyl ghrelin is unknown because it does not bind to the GHS-R 1a. Particularly novel is that the n-octanoyl moiety is an indispensable determinant of the GHS 1a receptor conformation. Although n-octanoyl ghrelin is in only low amounts, it is located in neurons of hypothalamic nuclei involved in the regulation of food intake, especially the arcuate neurons. In addition, it is now possible to conclude the yet-to-be characterized highly specific acyl transferase enzyme that covalently links the n-octanoyl moiety to ghrelin also exists in the neurons because octanoylation of ghrelin probably occurs only intracellularly concomitant with biosynthesis of ghrelin and not extracellularly. Because of the cellular location of n-octanoyl ghrelin in the hypothalamus, results obtained thus far relate to food intake and not to GH secretion. The second major achievement of the authors, which evolved from acute food intake and chronic fasting studies in rats, reveals hormonal and molecular changes that coordinately occur in the hypothalamus-stomach-plasma during caloric restriction and forecasts new insight into how the ghrelin system may be involved in regulation of food intake. From a more general viewpoint, hypothalamic n-octanoyl ghrelin now must be incorporated as a functional component of the ghrelin system.
In previous publications, the existence of hypothalamic ghrelin has been inconsistently reported and, until the present study, never completely validated as the bioactive molecular form of n-octanoyl ghrelin. Additionally, the authors, using highly validated assays including processing of plasma and established reference standards, demonstrate the remarkable constant des-octanoyl:n-octanoyl ghrelin ratio in tissue (hypothalamus and stomach) and plasma under three experimental conditions. Higher plasma des-octanoyl ghrelin levels that originate at sites peripheral to the stomach probably involve different mechanisms such as removal of the octanoyl moiety in plasma, different pharmacokinetics of the two molecular forms of ghrelin, and possibly even secretion of only des-octanoyl ghrelin into the blood from select tissue sites other than the stomach because the octanoylating enzyme may not be present at these sites. These issues are one reason plasma total ghrelin levels are so difficult to interpret.
The precision of the des-octanoyl:n-octanoyl ratio (1.8:1) obtained for the hypothalamus and stomach tissue is speculated to originate initially at the level of biosynthesis of n-octanoyl ghrelin. Presumably, the 117 preproghrelin molecule is n-octanoyl modified by a highly specific acyl transferase enzyme during preproghrelin biosynthesis or posttranslationally before intracellular conversion to n-octanoyl ghrelin and des-octanoyl ghrelin in stoichiometric amounts. The precision and marked differences in the ratios of these two molecular forms for tissue vs. plasma appear to be revealing new knowledge about the function of the ghrelin system.
Of major significance is the strong evidence by the authors in 24- and 48-h fasting studies, which supports that the gene expression and production of n-octanoyl ghrelin are different in pattern implying the hypothalamus-stomach-plasma ghrelin system functions differently than currently perceived. In this study, hypothalamic n-octanoyl ghrelin mRNA and content decreased and stomach mRNA increased with decreased content, whereas plasma levels and the three hypothalamic orexigenic neuropeptides (neuropeptide Y, agouti-related protein, melanin-concentrating hormone), increased. Unexpected and seemingly still unexplained is that hypothalamic ghrelin synthesis and content decreased, which, from results of previous studies, would be expected to be increased during fasting and increased food intake. n-Octanoyl ghrelin is well known to increase food intake via its hypothalamic action and increase of the three neuropeptides. The authors’ interpretation of these results was that the decrease in hypothalamic n-octanoyl ghrelin content and production reflects increased release, but this would not explain the consistent concomitant decrease in hypothalamic ghrelin synthesis. Understanding these results is likely to reveal new insight about the regulation of the ghrelin system at the hypothalamic level.
In the 2-h study that was performed by administering 2-deoxy-D-glucose to block glucose cellular utilization, data were obtained that in part overlap with that obtained in longer 24- to 48-h fasting studies. The important notable results of the 2-h study were the marked increase of food intake with an associated decrease of hypothalamic ghrelin mRNA and content and an increase of the three orexigenic hypothalamic neuropeptides (neuropeptide Y, agouti-related protein, melanin-concentrating hormone) without changes in stomach ghrelin mRNA, content, or plasma n-octanoyl ghrelin levels. One possible explanation for the paradoxical decrease of hypothalamic n-octanoyl ghrelin in the 2-h acute study and also in the 24- to 48-h chronic fasted rat studies is that it may play a sensory regulatory role, whereas stomach n-octanoyl ghrelin has a stimulatory role in the regulation of food intake. Thus, as caloric restriction occurs, less hypothalamic ghrelin GHS-R 1a is occupied and the receptor becomes more sensitive to the actions of circulating n-octanoyl ghrelin originating from the stomach. Even when the circulatory level of n-octanoyl ghrelin remained unchanged as in the 2-deoxy-D-glucose rat study and hypothalamic n-octanoyl ghrelin was decreased, food intake and the hypothalamic neuropeptides were increased, presumably because of enhanced sensitivity of the ghrelin GHS-R 1a to circulating plasma octanoyl ghrelin of stomach origin. Also, enhanced sensitivity of the stomach n-octanoyl ghrelin action may be increased as a result of increased receptor number. It seems reasonable even to consider that decreased hypothalamic ghrelin synthesis and production may be a regulatory sensor for food hunger, but this would not explain the paradoxical results observed of decreased hypothalamic n-octanoyl ghrelin in conjunction with a dramatic increase of food intake and the three neuropeptides. Also, it is remotely possible that the ghrelin system is only indirectly involved.
Already, there are a number of studies that could be directly/indirectly related to the role of hypothalamic n-octanoyl ghrelin. In particular, a characteristic of the GHS-R 1a has been its high in vitro constitutive signaling activity, raising the possibility that even when hypothalamic n-octanoyl ghrelin is decreased, the receptor signaling activity is elevated, which would be independent of stimulation (5). As Holst et al. (5) discussed, the constitutive signaling activity of the receptor is more difficult to demonstrate in vivo than in vitro; however, the ghrelin GHS-R 1a activity is very similar in magnitude to the activity of one of the most active constitutive receptors, ORF-74 oncogene, and thus this reflects an important physiological in vivo phenomenon. It has been previously reported by Bitar et al. (6) that [DArg1, DPhe5, DTrp7,9, Leu11]-Substance P is a competitive GH-releasing peptide (GHRP) GHS-R 1a antagonist of low potency; however, as revealed by Holst et al. (5), it is a potent inverse agonist that inhibits the constitutive signaling activity of the GHS-R 1a in an unstimulated state, and thus they proposed inverse agonists may become valuable in the treatment of obesity. Almost all evidence strongly supports the importance of the GHS-R 1a because it mediates the ghrelin and GHRP/GHS action on both GH and food intake. Also notable is that food intake and GH secretion are increased by ghrelin/GHRP in humans and, in rats, both these effects are stimulated by ghrelin/GHRP and inhibited by GHRP receptor antagonists (1, 2, 7, 8). Additionally, when rats were fasted for 48 h, the hypothalamic ghrelin GHS-R1a mRNA levels rose 8-fold, indicating a marked increase in gene expression that would be expected to increase sensitivity to and effects of n-octanoyl ghrelin (9).
Two reasons for being enthusiastic about these studies are that n-octanoyl ghrelin is the only known peripheral orexigenic activator that functions in a complex multicomponent system via the hypothalamus-stomach-plasma and because these data forecast new dimensions of how the ghrelin system functions. Complete validation of the existence of hypothalamic n-octanoyl ghrelin is envisioned to have a significant fundamental and clinical impact.
References
van der Lely AJ, Tsch?p M, Heiman M, Ghigo E 2004 Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 25:426–457
Korbonits M, Goldstone AP, Gueorguiev M, Grossman AB 2004 Ghrelin-a hormone with multiple functions. Front Neuroendocrinol 25:27–68
Inui A, Asakawa A, Bowers CY, Mantovani G, Laviano A, Meguid MM, Fujimiya M 2004 Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ. FASEB J 18:439–456
Sato T, Fukue Y, Teranishi H, Yoshida Y, Kojima M 2005 Molecular forms of hypothalamic ghrelin and its regulation by fasting and 2-deoxy-D-glucose administration. Endocrinology 146:2510–2516
Holst B, Holliday ND, Bach A, Elling CE, Cox HM, Schwartz TW 2004 Common structural basis for constitutive activity of the ghrelin receptor family. J Biol Chem 51:53806–53817
Bitar KG, Bowers CY, Coy DH 1991 Effects of substance P/bombesin antagonists on the release of growth hormone by GHRP and GHRH. Biochem Biophys Res Commun 180:156–161
Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR 2001 Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 86:5992–5995
Laferrère B, Abraham C, Russell CD, Bowers CY 2005 Growth hormone releasing peptide-2 (GHRP-2), like ghrelin, increases food intake in healthy men. J Clin Endocrinol Metab 90:611–614
Kim MS, Yoon CY, Park KH, Shin CS, Park KS, Kim SY, Cho BY, Lee HK 2003 Changes in ghrelin and ghrelin receptor expression according to feeding status. Neuroreport 14:1317–1320(Cyril Y. Bowers)
Address all correspondence and requests for reprints to: Cyril Y. Bowers, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL53, New Orleans, Louisiana 70112. E-mail: cybowers@tulane.edu.
n-Octanoyl ghrelin continues to be noted for its stimulatory action on the GHS-R 1a [GH secretagogue (GHS) receptor] and regulation of GH secretion and food intake (1, 2, 3). This now has been extended more in depth in regard to regulation of food intake as revealed in the publication of Sato and Kojima and co-workers (4). The high-quality, validated approaches and techniques used by the authors to quantitate the molecular forms of hypothalamic ghrelin in vivo in rats is the first reason for concluding that the study is of major importance. At last, unequivocal in vivo evidence has been obtained that n-octanoyl ghrelin exists and is synthesized in the hypothalamus, and that it and des-acyl (des-octanoyl) ghrelin constitute the two major molecular forms of ghrelin in the hypothalamus. The role of des-octanoyl ghrelin is unknown because it does not bind to the GHS-R 1a. Particularly novel is that the n-octanoyl moiety is an indispensable determinant of the GHS 1a receptor conformation. Although n-octanoyl ghrelin is in only low amounts, it is located in neurons of hypothalamic nuclei involved in the regulation of food intake, especially the arcuate neurons. In addition, it is now possible to conclude the yet-to-be characterized highly specific acyl transferase enzyme that covalently links the n-octanoyl moiety to ghrelin also exists in the neurons because octanoylation of ghrelin probably occurs only intracellularly concomitant with biosynthesis of ghrelin and not extracellularly. Because of the cellular location of n-octanoyl ghrelin in the hypothalamus, results obtained thus far relate to food intake and not to GH secretion. The second major achievement of the authors, which evolved from acute food intake and chronic fasting studies in rats, reveals hormonal and molecular changes that coordinately occur in the hypothalamus-stomach-plasma during caloric restriction and forecasts new insight into how the ghrelin system may be involved in regulation of food intake. From a more general viewpoint, hypothalamic n-octanoyl ghrelin now must be incorporated as a functional component of the ghrelin system.
In previous publications, the existence of hypothalamic ghrelin has been inconsistently reported and, until the present study, never completely validated as the bioactive molecular form of n-octanoyl ghrelin. Additionally, the authors, using highly validated assays including processing of plasma and established reference standards, demonstrate the remarkable constant des-octanoyl:n-octanoyl ghrelin ratio in tissue (hypothalamus and stomach) and plasma under three experimental conditions. Higher plasma des-octanoyl ghrelin levels that originate at sites peripheral to the stomach probably involve different mechanisms such as removal of the octanoyl moiety in plasma, different pharmacokinetics of the two molecular forms of ghrelin, and possibly even secretion of only des-octanoyl ghrelin into the blood from select tissue sites other than the stomach because the octanoylating enzyme may not be present at these sites. These issues are one reason plasma total ghrelin levels are so difficult to interpret.
The precision of the des-octanoyl:n-octanoyl ratio (1.8:1) obtained for the hypothalamus and stomach tissue is speculated to originate initially at the level of biosynthesis of n-octanoyl ghrelin. Presumably, the 117 preproghrelin molecule is n-octanoyl modified by a highly specific acyl transferase enzyme during preproghrelin biosynthesis or posttranslationally before intracellular conversion to n-octanoyl ghrelin and des-octanoyl ghrelin in stoichiometric amounts. The precision and marked differences in the ratios of these two molecular forms for tissue vs. plasma appear to be revealing new knowledge about the function of the ghrelin system.
Of major significance is the strong evidence by the authors in 24- and 48-h fasting studies, which supports that the gene expression and production of n-octanoyl ghrelin are different in pattern implying the hypothalamus-stomach-plasma ghrelin system functions differently than currently perceived. In this study, hypothalamic n-octanoyl ghrelin mRNA and content decreased and stomach mRNA increased with decreased content, whereas plasma levels and the three hypothalamic orexigenic neuropeptides (neuropeptide Y, agouti-related protein, melanin-concentrating hormone), increased. Unexpected and seemingly still unexplained is that hypothalamic ghrelin synthesis and content decreased, which, from results of previous studies, would be expected to be increased during fasting and increased food intake. n-Octanoyl ghrelin is well known to increase food intake via its hypothalamic action and increase of the three neuropeptides. The authors’ interpretation of these results was that the decrease in hypothalamic n-octanoyl ghrelin content and production reflects increased release, but this would not explain the consistent concomitant decrease in hypothalamic ghrelin synthesis. Understanding these results is likely to reveal new insight about the regulation of the ghrelin system at the hypothalamic level.
In the 2-h study that was performed by administering 2-deoxy-D-glucose to block glucose cellular utilization, data were obtained that in part overlap with that obtained in longer 24- to 48-h fasting studies. The important notable results of the 2-h study were the marked increase of food intake with an associated decrease of hypothalamic ghrelin mRNA and content and an increase of the three orexigenic hypothalamic neuropeptides (neuropeptide Y, agouti-related protein, melanin-concentrating hormone) without changes in stomach ghrelin mRNA, content, or plasma n-octanoyl ghrelin levels. One possible explanation for the paradoxical decrease of hypothalamic n-octanoyl ghrelin in the 2-h acute study and also in the 24- to 48-h chronic fasted rat studies is that it may play a sensory regulatory role, whereas stomach n-octanoyl ghrelin has a stimulatory role in the regulation of food intake. Thus, as caloric restriction occurs, less hypothalamic ghrelin GHS-R 1a is occupied and the receptor becomes more sensitive to the actions of circulating n-octanoyl ghrelin originating from the stomach. Even when the circulatory level of n-octanoyl ghrelin remained unchanged as in the 2-deoxy-D-glucose rat study and hypothalamic n-octanoyl ghrelin was decreased, food intake and the hypothalamic neuropeptides were increased, presumably because of enhanced sensitivity of the ghrelin GHS-R 1a to circulating plasma octanoyl ghrelin of stomach origin. Also, enhanced sensitivity of the stomach n-octanoyl ghrelin action may be increased as a result of increased receptor number. It seems reasonable even to consider that decreased hypothalamic ghrelin synthesis and production may be a regulatory sensor for food hunger, but this would not explain the paradoxical results observed of decreased hypothalamic n-octanoyl ghrelin in conjunction with a dramatic increase of food intake and the three neuropeptides. Also, it is remotely possible that the ghrelin system is only indirectly involved.
Already, there are a number of studies that could be directly/indirectly related to the role of hypothalamic n-octanoyl ghrelin. In particular, a characteristic of the GHS-R 1a has been its high in vitro constitutive signaling activity, raising the possibility that even when hypothalamic n-octanoyl ghrelin is decreased, the receptor signaling activity is elevated, which would be independent of stimulation (5). As Holst et al. (5) discussed, the constitutive signaling activity of the receptor is more difficult to demonstrate in vivo than in vitro; however, the ghrelin GHS-R 1a activity is very similar in magnitude to the activity of one of the most active constitutive receptors, ORF-74 oncogene, and thus this reflects an important physiological in vivo phenomenon. It has been previously reported by Bitar et al. (6) that [DArg1, DPhe5, DTrp7,9, Leu11]-Substance P is a competitive GH-releasing peptide (GHRP) GHS-R 1a antagonist of low potency; however, as revealed by Holst et al. (5), it is a potent inverse agonist that inhibits the constitutive signaling activity of the GHS-R 1a in an unstimulated state, and thus they proposed inverse agonists may become valuable in the treatment of obesity. Almost all evidence strongly supports the importance of the GHS-R 1a because it mediates the ghrelin and GHRP/GHS action on both GH and food intake. Also notable is that food intake and GH secretion are increased by ghrelin/GHRP in humans and, in rats, both these effects are stimulated by ghrelin/GHRP and inhibited by GHRP receptor antagonists (1, 2, 7, 8). Additionally, when rats were fasted for 48 h, the hypothalamic ghrelin GHS-R1a mRNA levels rose 8-fold, indicating a marked increase in gene expression that would be expected to increase sensitivity to and effects of n-octanoyl ghrelin (9).
Two reasons for being enthusiastic about these studies are that n-octanoyl ghrelin is the only known peripheral orexigenic activator that functions in a complex multicomponent system via the hypothalamus-stomach-plasma and because these data forecast new dimensions of how the ghrelin system functions. Complete validation of the existence of hypothalamic n-octanoyl ghrelin is envisioned to have a significant fundamental and clinical impact.
References
van der Lely AJ, Tsch?p M, Heiman M, Ghigo E 2004 Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 25:426–457
Korbonits M, Goldstone AP, Gueorguiev M, Grossman AB 2004 Ghrelin-a hormone with multiple functions. Front Neuroendocrinol 25:27–68
Inui A, Asakawa A, Bowers CY, Mantovani G, Laviano A, Meguid MM, Fujimiya M 2004 Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ. FASEB J 18:439–456
Sato T, Fukue Y, Teranishi H, Yoshida Y, Kojima M 2005 Molecular forms of hypothalamic ghrelin and its regulation by fasting and 2-deoxy-D-glucose administration. Endocrinology 146:2510–2516
Holst B, Holliday ND, Bach A, Elling CE, Cox HM, Schwartz TW 2004 Common structural basis for constitutive activity of the ghrelin receptor family. J Biol Chem 51:53806–53817
Bitar KG, Bowers CY, Coy DH 1991 Effects of substance P/bombesin antagonists on the release of growth hormone by GHRP and GHRH. Biochem Biophys Res Commun 180:156–161
Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, Bloom SR 2001 Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol Metab 86:5992–5995
Laferrère B, Abraham C, Russell CD, Bowers CY 2005 Growth hormone releasing peptide-2 (GHRP-2), like ghrelin, increases food intake in healthy men. J Clin Endocrinol Metab 90:611–614
Kim MS, Yoon CY, Park KH, Shin CS, Park KS, Kim SY, Cho BY, Lee HK 2003 Changes in ghrelin and ghrelin receptor expression according to feeding status. Neuroreport 14:1317–1320(Cyril Y. Bowers)